Sulfatase (sulfuric ester hydrolase) has an activity to hydrolyze various biomolecules containing ester-linked sulfate groups, releasing the sulfate groups. In a human, at least nine types of sulfatases occur which differ in their substrate specificity. Each of these sulfatases contains a formylglycine residue (2-amino-3-oxopropionate residue) in its peptide chain (Non-patent Document 1). This formylglycine residue, which is one of the amino acid residues constituting the active center, is a residue generated by conversion of a certain cysteine residue originally present in the amino acid sequence of the sulfatase just after its translation. The formylglycine residue is hydrated and occurs in a gem-diol form in the catalytic reactions of sulfatase, and one of the two hydroxyl groups of the gem-diol is essential for the generation of an enzyme-sulfuric acid ester intermediate, and the other hydroxyl group is required for separation of a sulfate group. Therefore, the conversion of the cysteine residue to a formylglycine residue is essential for the sulfatase activity. In multiple sulfatase deficiency, which is a genetic disease caused by severe reduction of all sulfatase activities, there is no abnormality in the sulfatase gene itself, but the normal conversion of the cysteine residue to a formylglycine residue fails to take place, and as a result, sulfatase activities are lost or markedly reduced (Non-patent Document 1, Patent Document 1).
Thus, in quantitative determination of a sulfatase in a living body, measurement of mere its total amount is insufficient for quantitative evaluation of its enzyme activity, and thus it is necessary to determine the ratio at which the cysteine residue has been converted into a formylglycine residue in the sulfatase. The same is true of a recombinant sulfatase manufactured using recombinant DNA technology. As for a recombinant sulfatase, a method for production of arylsulfatase A has been reported, in which the cysteine residue has been converted into a formylglycine residue at a desired ratio (Patent Document 2). With an enzyme produced by this method, too, the ratio must be determined at which the cysteine residue has been converted into a formylglycine residue.
Iduronate 2-sulfatase (I2S) is one of sulfatases having an activity to hydrolyze sulfate ester bonds of heparan sulfate and dermatan sulfate, both belonging to the glycosaminoglycans. In order for I2S to exhibit its enzymatic activity, it is also necessary, like other sulfatases, that its predetermined cysteine residue located in the active center has been converted into a formylglycine residue.
Genetic deficiency of this enzyme leads to the development of Hunter syndrome (mucopolysaccharidosis type II), associated with such signs as skeletal abnormalities, caused by abnormal metabolism of heparan sulfate and dermatan sulfate and resulting accumulation of their partial degradation products in the tissues such as the liver and spleen. For patients with Hunter syndrome, enzyme replacement therapy is performed to supplement I2S. I2S employed in enzyme replacement therapy for Hunter syndrome has been produced as recombinant human I2S using CHO cells transformed with an expression vector with an incorporated human I2S gene. Various methods for producing recombinant human I2S using CHO cells have been reported (Patent Documents 3 and 4).
In order for a recombinant human I2S to exhibit its enzymatic activity, it is also necessary, like naturally occurring I2S, that the cysteine residue has been converted into a formylglycine residue. Thus, in quantitative determination of a recombinant human I2S, measurement of mere its total amount is insufficient for quantitative evaluation of its enzyme activity, and thus it is necessary to determine the ratio at which the cysteine residue has been converted into a formylglycine residue in the I2S.
N-acetylgalactosamine-4-sulfatase (ASB), also called arylsulfatase B, is one of sulfatases and has an activity to release sulfuric acid ions by hydrolyzing chondroitin-4-sulphate, dermatan sulfate and UDP-N-acetylgalactosamine-4-sulfate. In order for ASB to exhibit its enzymatic activity, it is also necessary, like other sulfatases, that its predetermined cysteine residue located in the active center has been converted into a formylglycine residue. Genetical deficiency of this enzyme would cause accumulation of dermatan sulfate and the like in the lysosomes of a wide range of tissues, which results in the development of Maroteaux-Lamy syndrome (mucopolys accharidosis type VI), which exhibits such symptoms as growth retardation, marked deformation of the spine and limbs, hepatosplenomegaly, and congenital cataract. For patients with Maroteaux-Lamy syndrome, enzyme replacement therapy is performed to supplement ASB. ASB used in enzyme replacement therapy of Maroteaux-Lamy syndrome has been produced as a recombinant human ASB using CHO cells transformed with an expression vector with an incorporated human ASB gene (Patent Document 5).
In order for a recombinant human ASB to exhibit its enzymatic activity, it is also necessary, like naturally occurring ASB, that the cysteine residue has been converted into a formylglycine residue. Thus, in quantitative determination of a recombinant human ASB, measurement of mere its total amount is insufficient for quantitative evaluation of its enzyme activity, and it is necessary to determine the ratio at which the cysteine residue has been converted into a formylglycine residue in the ABS.
In addition to Hunter syndrome and Maroteaux-Lamy syndrome, the diseases caused by deficiency of sulfatase include Morquio disease type A, and San Filippo syndrome A and D types, in which are found genetic deficiency of N acetylgalactosamine-6-sulfatase, heparan-N-sulfatase, and N-acetyl glucosamine-6-sulfate sulfatase, respectively. As for treatment of these diseases also, application of enzyme replacement therapy is conceivable employing enzymes produced using recombinant technology, in which, too, conversion of the cysteine residue to a formylglycine residue is essential in order for these enzymes produced using recombinant technology to exhibit their enzymatic activities. Thus, in quantitative determination of each of these enzymes, measurement of mere its total amount is insufficient, and it is necessary to determine the ratio at which the cysteine residue has been converted into a formylglycine residue in the enzymes.
As a method for determination of the amount of a sulfatase in which the particular cysteine residue originally present in the sulfatase is converted into a formylglycine residue, there is known a method comprising; digesting the sulfatase into peptide fragments by trypsin treatment, and then subjecting the peptide fragments to reverse phase column chromatography, and comparing, on the chromatogram thus produced, a peak corresponding to a peptide fragment containing the formylglycine residue with a peak corresponding to a peptide fragment containing the cysteine residue (Non-patent Document 1). This method does not allow one to identify, on a resulting chromatogram alone, the peak corresponding to a cysteine residue-containing peptide fragment or to a formylglycine residue-containing peptide fragment. According to this method, therefore, it is required to collect all the fractions corresponding to respective peaks, and then analyze the amino acid sequence of the peptide fragment contained in each of these fractions, one by one, to identify the aimed peptide fragments. Thus, it is very complicated to follow its procedure. And in the case of a protein made up of a long peptide chain, its treatment with trypsin gives an increased number of peptide fragments, and so an increased number of peaks are produced by their separation with reverse phase column chromatography. This, therefore, makes the procedure more complicated.